During the course of gene transfer experiments into K562 cells using a modified adenovirus vector, we noted that control K562 cell DNA showed a positive signal when probed with the entire adenovirus genome. This signal was also present in all normal human genomic DNA tested. The adenovirus genome was resolved into three fragments which, when used as probes in Southern blot analysis showed evidence for homology between the 5'9 kb of adenovirus and a 2.5 kb Sst I fragment of human DNA. Because the 5' adenovirus fragment contains coding sequences for proteins involved in DNA replication, transcriptional control, and cell transformation, we chose to clone the human sequence homologous to the fragment. In cloning this sequence out of a human cosmid library, we identified a moderately repetitive DNA sequence family consisting of tandem arrays of 2.5 kb members. A member was sequenced and several non-adjacent, 15-20 bp G-C rich segments with homology to the left side of adenovirus were discovered. The copy number of 400 members is highly conserved among humans. Southern blots of partial digest of human DNA have verified the tandem array of the sequence family. The chromosomal location was defined by somatic cell genetics and in situ hybridization. Tandem arrays are found only on chromosomes 4 (q31) and 19 (q13.1-q13.3). Homologous repetitive sequences are found in DNA of other primates but not in cat or mouse. Thus we have identified a new family of moderately repetitive DNA sequences, unique because of its organization in clustered tandem arrays, its length, its chromosomal location, and its lack of homology to other moderately repetitive sequence families.